Project Summary. While rates of antibiotic resistance bacterial infections continue to rise, our development of new antimicrobials has stagnated. The failure of traditional small molecule leads to restock are arsenal demands the development of alternative approaches to prevent our slide back to the pre-antibiotic medical era. Antimicrobial peptides are highly effective against multidrug resistant pathogens but have poor pharmacological properties hindering their in vivo use. Antibodies have excellent pharmacological properties but generally lack direct antimicrobial action, which limits their therapeutic use in treating infection, especially in immune compromised populations most at risk for multidrug resistant infection. The objective of this proposal is to develop a new therapeutic paradigm that joins the direct action of antimicrobial peptide antibiotics with the benefits of antibody structure in a single antimicrobial agent. To develop this new modality we are leveraging the unique qualities of variable heavy chain domain of Camelid heavy chain antibodies, known as a VHH or nanobody. Supported by a high- throughput approaches for lead antimicrobial nanobody identification we have developed, this new paradigm takes advantage of the benefit of direct antimicrobial peptide activity while providing a modular way to multiplex peptide action in a pharmacologically beneficial nanobody backbone structure. Our approach has the ability to identify both broad spectrum and target antimicrobials to selectively eliminate invading pathogens. Furthermore, our approach has the future potential to utilize immune functions for enhanced pathogen clearance. This proposal offers a quantum leap forward in anti-infective research. Completion of the planned work is expected to have a positive translational impact by introducing a new direction in immune based anti- infective therapy and new high-throughput tools for lead antimicrobial nanobody discovery, both of which will support the fight against antibiotic resistant bacteria. ! !